A barley gene encoding a novel DNA-binding protein (HRT) was identified by southwestern screening with baits containing a gibberellin phytohormone response element from an alpha-amylase promoter. The HRT gene contains two introns, the larger of which (5722 base pairs (bp)) contains a 3094-bp LINE-like element with homology to maize Colonist1. In vitro mutagenesis and zinc- and DNA-binding assays demonstrate that HRT contains three unusual zinc fingers with a CX8-9CX10CX2H consensus sequence. HRT is targeted to nuclei, and homologues are expressed in other plants. In vivo, functional tests in plant cells indicate that full-length HRT can repress expression from certain promoters including the Amy1/6-4 and Amy2/32 alpha-amylase promoters. In contrast, truncated forms of HRT containing DNA-binding domains can activate, or derepress, transcription from these promoters. Northern hybridizations indicate that HRT mRNA accumulates to low levels in various tissues. Roles for HRT in mediating developmental and phytohormone-responsive gene expression are discussed.